br An enzyme linked immunosorbent assay ELISA br
2.4. An enzyme-linked immunosorbent assay (ELISA)
Splenocytes from mice were seeded in flat-bottom 24-well plates (106 cells/well) in the presence of a plate-bound anti-CD3 antibody, and the supernatant was collected after 48 h. The supernatant concentra-tions of IFN-γ, TNF-α, IL-4, IL-13, and IL-10 in each sample were esti-mated by an ELISA.
The 96-well plates were then coated (overnight at 4 °C) with the capture antibody. Each well was washed three times with 0.05% Tween 20 in PBS (PBS-T), incubated with the blocking solution for 1 h at room temperature, and then washed four times with PBS-T. The samples and diluted standards were added, and the plate was incubated overnight at 4 °C. After four wash cycles, the detection antibody was added. Following 45 min of incubation at room temperature, the wells were washed. Avidin-conjugated alkaline phosphatase was added, and the plates were incubated at room temperature for 30 min. Immediately
Fig. 2. Cytotoxicity of extracts toward Panc02 FLAG Peptide and dose determination for an in vivo assay. A variety of concentrations of NL and TL extracts were applied to confirm the cytotoxicity toward Panc02 cells by the MTT assay (A). Cell viability was normalized to that in the vehicle-treated group. IC50 values of NL and TL treatment were determined: 1.7 and 0.3 mg/mL, respectively. For an in vivo assay, three concentrations (20, 40, or 80 mg/mL) were injected intraperitoneally daily for 3 days in a row into C57BL/6 male mice, but no significant changes in body weight or clinical signs were detected compared to non-treated group (B–D). The concentration 80 mg/mL was employed for further in vivo experiments. Data are presented as the means ± SD from three independent experiments (Non-treatment: non-treated group; NL: NL treated group; TL: TL treated group).
after, a substrate solution was added, and the plates were kept at room temperature for 5–30 min before the addition of stop buﬀer. Absorbance was read at 405 nm on a microplate reader (Emax, Molecular Devices, San Jose, CA, USA).
2.5. Purification of CD4+ T cells and Treg diﬀerentiation
Spleens were excised from Foxp3-GFP mice, and CD4+ T cells were purified using a magnetic cell sorting system (MACS® separation, Miltenyi Biotech, Bergisch, North Rhine-Westphalia, Germany). Cells were cultured in the RPMI 1640 culture medium with 10% heat-in-activated FBS (Cellgro), 100 units/mL penicillin (Cellgro), 100 μg/mL streptomycin (Cellgro), 2 mM L-glutamine (Cellgro), and 2-mercap-toethanol (Sigma-Aldrich) at 37 °C in a 5% CO2 humidified incubator. Plate-bound anti-CD3 antibody (1 μg/mL, BD Bioscience) and anti-CD28 antibody (1 μg/mL, BD Bioscience) were used to stimulate the T cells. Recombinant mouse IL-2 (10 ng/mL, BD Bioscience) and TGF-β (2.5 ng/mL, BD Bioscience) were added to the culture to moderately induce Tregs.
2.6. Cancer cell isolation
To isolate cancer cells from a tumor tissue from its source, a tumor was minced into pieces in Hank’s balanced salt solution (HBSS, Sigma-Aldrich) and pulled into a 5 mL pipette without getting stuck. Tissue fragments were transferred to a nonvented tissue culture flask and di-gested with 1 mg/mL type IV collagenase, 20 U/mL type IV DNase, and 0.1 mg/mL type V hyaluronidase (Sigma-Aldrich) for 1–3 h with gentle shaking to dissociate the cells in reverse transcriptase buﬀer. Tumor cell suspensions were filtered through a 100 μm nylon mesh and then were washed in HBSS two times. Viability was determined by a trypan blue exclusion assay.
Splenocytes or cancer cells were collected into FACS buﬀer and stained with the following antibodies: a fluorescein isothiocyanate (FITC)-conjugated anti-Thy1.2 antibody, allophycocyanin (APC)-con-jugated anti-CD4, APC-conjugated anti-CD8, phycoerythrin (PE)-con-jugated anti-CD19, anti-CD45R, FITC-conjugated anti-CD11b, PE-con-jugated anti-F4/80, FITC-conjugated anti-CD11c, PE-conjugated anti-MHC class II, FITC-conjugated anti-NKp46, PE-conjugated anti-NK1.1, PE-conjugated anti-CD62L, and a FITC-conjugated anti-CD44 antibody (BD Bioscience and eBioscience, Waltham, MA, USA). The stained cells were analyzed by means of the FACSCalibur flow cytometer and Cell Quest analysis software (BD Bioscience).
2.8. Extraction and analysis of carotenoids
To concentrate carotenoids in both extracts, the method reported by Tuan et al. (2012) was slightly modified. Saponification, which is the essential step for collecting carotenoids, was performed by exposing both extracts to 10% KOH overnight. Both samples were then re-suspended in water followed by partitioning with n-hexane. Then, n-hexane layers were concentrated in vacuum and used as analytical samples. The analysis of carotenoids in samples was performed by high-performance liquid chromatography (HPLC) using a previously re-ported method (Tuan et al., 2012). An Agilent 1260 infinity LC system (Agilent Technologies, Santa Clara, CA, USA) and Prontosil 200-5-C30 column (Bischoﬀ Chromatography, Leonberg, Germany) were em-ployed for this analysis.